Flourescent aquatic bioassay and procedure

ABSTRACT

The present invention relates to a novel assay for determining levels of toxicants in aqueous environments, preferably in water supplies. The present invention also relates to a method for utilizing the assay to test the level of toxicants in an aquatic source. Further embodiments of the present invention relate to a test kit embodying the assay of the present invention. More specifically the assay involves the use of enzyme substrates having an umbelliferyl group and multi-cellular organisms having bodies which fluoresce.

This patent application is a continuation-in-part application of U.S.Ser. No. 343,360 entitled, "Fluorescent Aquatic Bioassay Procedures",filed Apr. 26, 1989, now abandoned,

FIELD OF THE INVENTION

The present invention relates to an aquatic environmental toxicologyassay employing fluorescent markers, a method of using this novel assayand a kit for use in aquatic analysis.

BACKGROUND OF THE INVENTION

There are a number of enzymatic activity tests which have usedspectrophotometric or fluoremetric methods to enumerate active andinactive microbes [See, for example, Flint, European J. Appl.Microbiol., 4, 195 (1977); Dutton, et al., Wat. Res., 20, 1461 (1986);Burton, et al., Wat. Res., 21, 1173 (1987); "Sediment Microbial ActivityTests for the Detection of Toxicant Impacts" Aquatic Toxicology: SeventhSymposium, ASTM STP R. D. Caldwell, et al., Ed. American Society forTesting and Materials, Philadelphia, 1985 pp. 214-228; EnvironmentalToxicology and Chemistry, 8, 1057 (1989); Toxicity Assessment, 4, 149(1989); Toxicity Assessment, 4, 255 (1989); Cloete, et al., Wat. Res.,22, 961 (1988); Wat. Res., 22, 971 (1988); Katayama-Hirayama, Wat. Res.,20, 491 (1986); Obst, Fresnius Z. Anal. Chem 321, 166, (1985); Obst, etal., Toxicity Assessment, 3, 81 (1988)]; and to determine active biomass[See, for example, Teuber, et al., European J. Appl. Microbiol., 4, 185(1977); Schnurer, Applied and Environmental Microbiology, 43, 1256(1982); Hoppe, Mar. Ecol. Prog. Ser., II, 299 (1983); and Tan, MarineBiology. 76, 247 (1983).

Fluorescent indicators have been utilized in cats and rats to measurecentral nervous system pH [See, for example, Sundt, et al.,"Umbelliferone as an Intracellular pH-Sensitive Fluorescent Indicatorand Blood-Brain Barrier Probe: Instrumentation, Calibration andAnalysis", 60 (1980)]. U.S. Pat. No. 4,534,317 discloses fluorescingdyes which are used to monitor fish food consumption.

None of the methodologies disclosed in the art utilizes a series of testconcentrations to develop standard LC50 and EC50 toxicity values thatare required by the Federal Water Pollution Control Act Amendments(Clean Water Act) of 1977 (PL 95-217). Section 101(a)(3). Nor do any ofthese methodologies provide a measure of toxicants in an aquatic body ina fast and efficient manner.

The present preferred method for determining aquatic toxicity in watersupplies generally utilizes a 48 hour daphnia test or a 96 hour fatheadminnow test. In these methodologies, the multicellular organisms areexposed to a toxicant for a period of 2 to 4 days and then the liveorganisms are counted to determine death rate. Other bioassay tests,which utilize 24 hour to 21-day tests, are described by the UnitedStates Environmental mental Protection Agency, ASTM, SETAC, OECD andvarious other State and private research groups. There is a clear needin the art for a fast, easy to use bioassay test which has reliabilityand accuracy, and which may be used with confidence by a lay person.

It is an object of the present invention to provide an aquatic bioassaythat can be used to measure various toxicants in aquatic sources in aneasy, fast and efficient manner.

It is a further object of the present invention to provide a novelaquatic bioassay which ca test both lethal (acute) and sublethal(chornic) concentrations of toxicants in aquatic sources.

It is still an additional object of the present invention to provide amethod for determining the concentrations of various toxicants inaquatic sources in a fast, efficient manner.

It is still another object of the present invention to provide abioassay kit for testing toxicants in aquatic sources.

These and other objects of the present invention may be readilydetermined by a review of the description and the examples of thepresent invention.

SUMMARY OF THE INVENTION

The present invention relates to a novel assay for determining levelsand effects of toxicants in aqueous environments, preferably in watersupplies. The present invention also relates to a method for utilizingthe assay to test the level of toxicants in an aquatic source. Furtherembodiments of the present invention relate to a test kit embodying theassay of the present invention.

In a first aspect, the present invention relates to an aquatic bioassayfor determining the existence of toxicants in an aquatic sourcecomprising:

a) at least one test sample comprising:

i. an aquatic source containing a toxicant to be tested;

ii. an effective amount of an enzyme substrate which results in afluorescent product after enzymatic modification; and

iii. a concentration of a living organism having an enzyme systemcapable of enzymatically modifying said enzyme substrate to produce saidfluorescent product in an amount sufficient to be fluorescentlyidentified; and

b) reference standards, at least one of said reference standardscomprising:

i. an aquatic source containing a known toxicant concentration;

ii. an identical amount of said enzyme substrate from (a)ii); and

identical concentration of said living organism from (a)iii);

and a second reference standard comprising;

i. an aquatic source containing an absence of toxicant;

ii. an identical amount of said enzyme substrate from (a)ii); and

iii. an identical concentration of said living organism from (a)iii).

In addition, the above bioassay preferably contains at least threereference standards, each reference standard containing differentconcentrations of toxicant.

In the bioassay of the present invention, any aquatic source may beanalyzed to determine the concentration or effect of toxicants. Sourcesof media from reservoirs, rivers, lakes, streams, ponds and other bodiesof water, industrial wastestreams, municipal wastestreams, leachate,elutriate, liquid phase, suspended particulate phase, point andnon-point source samples and pure compounds, and aqueous environments,including salt water aquatic sources may be tested to determine thelevels and effects of toxicants for purposes of meeting water safetyguidelines, to determine the safety of drinking water and fordetermining the effect of toxicants on the microorganisms in sludge atwaste treatment centers, among other uses.

The bioassay of the present invention utilizes a test sample containinga water source to be analyzed for a toxicant or toxicants and severalreference samples, each reference sample containing an amount of atoxicant which is different from another reference source. All thesamples, including the test sample and reference samples, preferablycontain substantially identical concentrations of enzyme substrate andnumbers of living organisms. In general, each test sample and referencesample is preferably analyzed in triplicate.

In the method of the present invention, a test sample is prepared bymeasuring out an aqueous test sample and placing a number of livingorganisms into the test sample. After an elapsed time period, a measuredamount of enzyme substrate is added to each concentration. Thisconcentration of enzyme substrate and number of living organisms issufficient to allow enzymatic modification of the enzyme substrate intoa fluorescent product which may be identified using standardfluoremetric techniques. In addition to the test sample, severalreference standards can be prepared (preferably, each in triplicate).Each reference standard contains a known concentration of toxicant whichmay range from zero to rather high concentrations. Each reference samplealso contains a concentration of enzyme substrate and living organismsidentical to that of the test sample.

After preparation of the test sample(s) and the reference standards, theenzyme substrate in a reference solvent, preferably distilled water, issonicated for a short period of time (generally, about 30 seconds toseveral minutes) and added to the test sample(s) and referencestandards, which are then incubated for a period of time sufficient toallow enzymatic modification of the enzyme substrate into a fluorescentproduct. This period of incubation generally ranges from about a minuteto about several hours. Generally, the incubation temperature willdepend upon the species of living organism used in the bioassay and thekinetics of the enzyme performing the modification of substrate.However, in general, the test samples and reference samples areincubated at a temperature ranging from about 0° C. to about 25° C.,although higher and lower temperatures may be used. After allowingincubation for a period of time sufficient to enable the enzyme tomodify the enzyme substrate, each sample is measured for fluorescence byexposing the chambers to an ultraviolet (UV) light source and thenobserving the fluorescence visually or alternatively with a fluorometer.After the fluorescence of each sample is determined, the fluorescence ofthe test sample is compared with the fluorescence of the referencestandards. This may be done by simple visualization or alternativelywith instrumentation, such as a fluorometer (Sequoia-Turno Model #450,Farrand Mark Spectrofluorometer or Gilford Fluoro IV ScanningSpectrofluorometer).

By comparing the intensity of the fluroescence of the test sample withthe reference standards, the amount of toxicant and/or the toxic effectof the tested aquatic source can be determined. Using this procedure theconcentration of one or more toxicants may be measured. In addition,standard LC₅₀ and EC₅₀ toxicity values for a broad range of toxicantsmay be easily predicted and/or determined.

The present invention also relates to a test kit comprising an enzymesubstrate in an amount sufficient to produce a final concentration ofabout 1 ppm to about 100 ppm and directions for using the enzymesubstrate. In certain kits, live (including encysted) organisms innumbers sufficient to produce fluorescent product in a measurable amountmay also be included. In addition to enzyme substrate or substrate andlive organisms the kit of the present invention may also contain any oneor more of the following: exposure chambers such as a multiwell plate ortest tubes, distilled water, pipettes (test media and diluent media),substrate mixing vial, sonicator, directions, black light (UV source),fluorometer, UV safety glasses or goggles, a light box, an autopipettor,safety gloves, score card, statistical programs, associated packaging, ablack box and combinations of these components.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graphic representation of the EC₅₀ values of Example 2correlated to 48 hour LC50 values.

FIG. 2 compares data generated from Example 2 with Microtox high and lowEC₅₀ pure compound values taken from the literature. In almost everycase, the present invention was as sensitive as or more sensitive thanthe Microtox tests.

FIG. 3 compares data generated from Example 2 with 96 hour LC₅₀ purecompound values taken from the literature for Pimephales promelas(fathead minnow). In this comparison, the present invention was assensitive as or more sensitive than the fathead minnow test.

FIG. 4 compares data generated when several complex effluents werebioassayed with conventional 48 hour LC₅₀ methodology and the presentinvention. The results produced an excellent correlation (R² valuesranged from 0.90 to 0.96). In this figure, the EC₅₀ and LC₅₀ values areexpressed in percent effluent.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are used throughout the specifiction todescribe the present invention.

The term "toxicant" is used throughout the specification to describe anytoxic substance in a water supply which may have a deleterious effect ona biological system and includes chemical compositions, macromolecules,biochemicals, ions and radioactive material.

The term "enzyme substrate" is used throughout the specification todescribe a substrate which will produce a fluorescent product uponmodification.

The term "living organism" is used throughout the specification todescribe an organism having an enzyme system that is capable ofmodifying the enzyme substrate of the present invention to produce ameasurable fluorescent product. The term living organism includes activeas well as dormant forms including encysted organisms.

The term "aquatic souce" is used throughout the specification todescribe a water source that is to be measured. The term aquatic sourceincludes all types of fresh and salt water supplies.

The term "LC₅₀ " as used herein describes a concentration of toxicantwhich produces 50% lethality in test organisms.

The term "EC₅₀ " as used herein describes a concentration of toxicantwhich produces a toxic affect and a measurable loss in the ability to ofthe tested organism to function, i.e., a loss in swimming ability, lossin reproductive ability, etc.

Numerous enzyme substrates may be used in the present invention andinclude any substrate which fluoresces after enyzmatic modification toproduce a fluorescent product. Enzyme substrates containing any moietywhich, when cleaved will result in a fluorescent product, may be used inthe present invention. However, it has been found that the umbelliferylgroup which produces umbelliferone is especially useful in embodimentsof the present invention. Enzyme substrates having an umbelliferyl groupfor use in the present invention include, for example,4-Methylumbelliferyl b-D-Galactoside, 4-Methylumbelliferyla-D-Glucoside, 4-Methylumbelliferyl b-D-Xyloside, 4-Methylumbelliferylacetate, 4-Methylumbelliferyl N-Acetyl-b-D-Galactosaminide,4-Methylumbelliferyl N-Acetyl-a-D-Glucosaminide,4-Methylumbelliferyl-b-D-Glucosaminide,4-Methylumbelliferyl-a-L-Arabinofuranoside,4-Methylumbelliferyl-a-L-Arabinoside, 4-Methylumbelliferyl Butyrate,4-Methylumbelliferyl b-D-Cellobioside, 4-Methylumbelliferylb-D-N,N'-Diacetyl-Chitobioside, 4-Methylumbelliferyl Elaidate,4-Methylumbelliferyl b-D-Fucoside, 4-Methylumbelliferyl a-L-Fucoside,4-Methylumbelliferyl b-L-Fucoside, 4-Methylumbelliferyl a-D-Galactoside,4-Methylumbelliferyl b-D-Glucoside, 4-Methylumbelliferylb-D-Glucuronide, 4-Methylumbelliferyl Heptanoate, 4-Methylumbelliferyla-D-Mannopyranoside, 4-Methylumbelliferyl b-D-Mannopyranoside,4-Methylumbelliferyl Oleate, 4-Methylumbelliferyl Palmitate,4-Methylumbelliferyl Phosphate, 4-Methylumbelliferyl Propionate,4-Methylumbelliferyl Stearate, 4-Methylumbelliferyl Sulfate,4-Methylumbelliferyl b-D-N,N',N'"-Triacetyl Chitotriose,4-Methylumbelliferyl 2,3,5-Tri-0-Benzoyl-a-L-Arabinofuranoside, amongothers.

The amount of enzyme substrate used in the present invention is thatamount which produces a fluorescent product upon enzymatic modification.It has been found useful to include an amount of at least one enzymesubstrate ranging from about 1 part per million (ppm) to about 100 partsper million. However, amounts outside these ranges may also be used.

The enzyme substrate may be presented directly in the test and referencesamples or alternatively, may be sonicated, milled or otherwisephysically modified before being used.

The bioassay of the present invention makes use of a living organism tocleave the fluoremetric markers from the enzyme substrate and produce afluorescent product. These fluorescent markers are cleaved from thesubstrate as a function of the viability of the test specie's enzymaticsystem. Virtually any living organism having an enzyme system which canmodify the fluorescent enzyme substrates to produce a fluorescentproduct may be used in the present invention. Typical enzymes or enzymesystems that may be useful for modifying the enzyme substrates toproduce fluorescent products according to the present invention include,for example, AMP Deaminase, Aryl hydrocarbon Hydroxylase,7-Ethoxycoumarin 0-Deethylase, Cytochrome P-450 (general activity),7-Alkoxycoumarin 0-Dealkylase, 0-Deethylase and Adenylate Cyclase, amongothers.

Useful living organisms for use in the present invention include, forexample, various bacteria, fungi, protozoans, Cnidaria, especially forexample, Hydra, members of the phylum Platyhelminthes, such asPlanarians, Nemertines, Aschelminthes, especially including rotifers,Brachiopods, such as Ligoula spp., various molluscs, especiallyincluding snails, clams, squid and octupi after feeding enzyme substrateincorporated into composite feed, Polychaetes, especially includingNereis spp., members of the phylum Arthropoda, especially includingmysid shrimp, insect larvae and Daphnia spp. and various vertebrates,including fish such as fathead minnows, amphibians and other groups invarious stages of their life cycles.

Especially useful organisms for use in the present invention includeDaphnia magna, Ceriodaphnia dubia, Daphnia pulex, and species of mixedbacterial and microbial cultures from municipal sewage and industrialwaste treatment plants, including Nitrosomonas spp. and Nitrobacter spp.In addition to the above living organisms, additional organisms that areespecially useful in the bioassay of the present invention include thoseorganisms whose bodies fluoresce after exposure to the fluorescentenzyme substrates. These organisms include, in addition to Daphnia spp.,Brachionus spp. (rotifers), Artemia salina (brine shrimp), juvenile andadult Pimephales promelas (fathead minnows) and fruit fly larvae, amongothers.

The number of organisms that are used in the samples of the presentinvention ranges greatly depending upon the type of organism utilized.For example, in the case of bacteria and other microbes, the number oforganisms will generally number in the millions, while in the case ofmulti-cellular organisms such as the Daphnia or fathead minnow, thenumber of organisms will generally be less than ten, preferably about 5organisms.

In determining the amount of toxicant in an aquatic source, thesensitivity of the bioassay ranges from parts per hundred (effluent froma factory) to parts per trillion (such as measuring the amount of aparticular ion or chemical compound such as 2,3,7,8-TCDD).

In general, in determining toxicant levels each sample, includingreference standard, is run in triplicate. The tests are generally runfor periods ranging from about 10 minutes to several hours dependingupon the organism and enzyme substrate used and the toxicant measured.

Typical United States Environmental Protection Agency (EPA) aquatictoxicity protocols require an aquatic toxicologist to observe the lethaland sublethal effects of toxicants for predetermined time intervals(i.e., 0, 4, 8, 24, 72 and 96 hours). To perform the EPA protocol, thefollowing steps are completed:

[a] develop a series of test concentrations of the aquatic source to betested;

[b] assign the test organisms to each test concentration; and

[c] observe the test for lethal and sublethal affects at predeterminedtime intervals.

The bioassay of the present invention simplifies the task of observinglethal and sublethal effects of toxicants on living organisms. Ratherthan having to perform the test over a period or one, two, four or moredays as in the case of the EPA test, the bioassay of the presentinvention allows the investigator to make an accurate determination oftoxicant levels in periods of as little as several minutes and generallywithin an hour or two.

In addition to the above, the bioassay and the methodology of thepresent invention allows a determination of the general health of anaquatic population. This would have significant utility at a fishhatchery to determine the relative health of the fish population. Thesubstrate could be delivered with feed or added directly to the watercolumn in a hatchery. In addition, the present invention could be usedto get a more complete picture of organism health, for example at ahatchery or in a water body. The toxicity test could also be used in awastetreatment plant to gauge what would be an allowable(non-deleterious) rate of introduction of a known toxicant into abiologically activated waste treatment plant. In addition, the presentinvention could be utilized to guage the effectiveness of a waste watertreatment plant's effectiveness.

The bioassay of the present invention may be used to measureconcentrations of a large number of toxicants. Basically, any toxicantwhich exhibits a deleterious affect in a living organism may be measuredaccurately including, for example copper, cadmium, mercury, zinc,chromium, phenol, benzene, formaldehyde, toluene, other organics,including polychlorinated benzene, sodium polychlorinated phenols andvarious salts, including sulfides and other toxicants.

In the method of the present invention, the procedure for determiningthe effects of a toxicant in an aquatic source on living organismsincludes the steps of:

1) preparing a test sample aquatic source, generally in triplicate;

2) preparing reference samples in triplicate, each triplicate set ofreference samples containing an amount of toxicant ranging from 0 up toat least about the LC₅₀ or EC₅₀ concentration for said toxicant;

3) adding living organisms to the samples from steps 1 and 2 in amountssufficient to produce a measurable fluorescent product from an enzymesubstrate incubated with said organisms and allowing a period ofincubation;

4) adding enzyme substrate to the samples from step 3 in quantitieseffective to produce a measurable fluorescent product and mixing thesamples to produce uniformity;

5) allowing a further period of incubation at a temperature ranging fromabout 0° C. to about 30° C. for a period of at least about one minute;and

6) exposing the samples to an ultraviolet light source and measuring thefluorescent light emitted.

Additional steps useful in the method of the present invention includesonicating the substrate solution and calculating LC₅₀ values and EC₅₀values using standard statistical methods. Alternatively, theEnvironmental Protection Agency (EPA) provides, on a regular basis,computer programs entitled "Movings Avergage", "Probit" or "Binomial"(available from the EPA), the general methodologies of which are readilyknown by those of ordinary skill in the art. The following examples areprovided for purposes of illustrating the present invention only and arenot to be taken as a limitation of the scope of the present invention.

EXAMPLE 1 Determination of an EC₅₀ Value in a Multiwell Plate

The objective of this example was to determine the EC₅₀ value and itsconfidence limits for the test substance copper sulfate (CuO₄) to thecommon water flea, Daphnia magna.

Using the invertebrate Daphnia magna as a test species a series of testconcentratins are established with the standard reference toxicantcopper sulfate, CuSo₄, in triplicate at the following concentrations:

    ______________________________________                                                0-A  ppm replicates 1, 2 and 3                                                0-B  ppm replicates 1, 2 and 3                                                0.01 ppm replicates 1, 2 and 3                                                0.1  ppm replicates 1, 2 and 3                                                1.0  ppm replicates 1, 2 and 3                                                10.0 ppm replicates 1, 2 and 3                                        ______________________________________                                    

One ml of each of the above CuSO₄ solutions was pippetted into amutiwell plate (3 replications per concentration, 1 ml per replicate).To each of 18 wells in the plate 5 Daphnia magna were added and exposedto the test concentrations for a period of five minutes. Then to each ofthe 18 wells, with the exception of 0-B 1, 2 and 3, 1.0 mg of4-Methylumbelliferyl b-D-Galactoside (MUF) was dropped onto the surfaceof each well. The powder settled to the bottom of each well. After 1minute of exposure, each sample was read. An ultra-violet light (blacklight) was held over the multiwell in a dark room. The fluorescentintensity of Controls 0-A ppm (maximum intensity) and Controls 0-B ppm(minimum intensity) were utilized as baselines to compare the effects oftest concentrations 0.01 through 10.0 ppm CuSO₄. The following results,set forth in Table 1, below were observed.

The EC₅₀ was determined to be 3.2 ppm.

                  TABLE 1                                                         ______________________________________                                                    Replicates                                                        Test Concentration                                                                          #1         #2       #3                                          ______________________________________                                        0-A ppm CuSO.sub.4                                                                          f          f        f                                                         all alive  all alive                                                                              all alive                                   0-B ppm CuSO.sub.4                                                                          f          f        f                                                         all alive  all alive                                                                              all alive                                   0.01 ppm CuSO.sub.4                                                                         f          f        f                                                         all alive  all alive                                                                              all alive                                   0.1 ppm CuSO.sub.4                                                                          f          f        f                                                         all alive  all alive                                                                              all alive                                   1.0 ppm CuSO.sub.4                                                                          f          f        f                                                         all alive  all alive                                                                              all alive                                   10.0 ppm CuSO.sub.4                                                                         nf         nf       nf                                                        all alive  all alive                                                                              all alive                                   ______________________________________                                         f = fluorescing                                                               nf = nonfluorescing                                                      

This test detected a sublethal adverse affect at 10.0 ppm CuSO₄ in lessthan 10 minutes.

EXAMPLE 2 Determination of the EC50 Value in Test Tubes

Using Daphnia magna as a test species a series of test concentrations ofthe standard reference toxicant CuSO₄ was established in triplicate.Each replicate consisted of 10 ml of test media in a 15 ml glass testtube. Five organisms were placed in each tube. The tubes were thenmaintained for a period of one hour at ambient room environmentalconditions. At the end of one hour 0.4 mg of the substrate4-Methylumbelliferyl b-D-galactoside (MUF) was added. Fifteen minutesafter the addition of the MUF the test tubes were taken into a darkroom. The control replicates were then observed under black light. Arecord was made of the number of strongly fluorescing Daphnia magnabodies. All bodies fluoresced strongly. These organisms were thencompared to each of the other triplicate concentrations. The number oforganisms that were fluorescing as strongly as the controls wererecorded.

The data was then analyzed statistically by conventional methods(Methods for Measuring the Acute Toxicity of Effluents to Freshwater andMarine Organisms (Third Edition) EPA/600/4-85/013). Each organism notfluorescing as strongly as the controls was treated as dead whenhandling the data. From this procedure EC₅₀ for Cu with Daphnia magnawas determined to be 0.23 ppm.

11 standard reference toxicants were then analyzed by the above method.The results of that analysis appear below in table 2.

                  TABLE 2                                                         ______________________________________                                        Compound   Mean EC50    Coefficient of Variation                              ______________________________________                                        Cu         0.23 ppm     17%                                                   NaPCP      1.0           8%                                                   Cd         0.41         11%                                                   NaLS       74.1         32%                                                   Phenol     37.1         19%                                                   Zn         4.3          11%                                                   Cr         3.7           7%                                                   Benzene    6.5           3%                                                   Hg         0.02         21%                                                   Formaldehyde                                                                             39.2           14.3%                                               Toluene    6.3          14%                                                   ______________________________________                                         Note: Three tests were conducted at different times for each toxicant.   

When the preceding EC₅₀ values were correlated to historical Daphniamagna 48 hour LC50 values, there is an excellent correlation (R² 32 0.81with a significance of 0.004). This data is set forth in FIG. 1. In thefigure, the diagonal line represents the "line of equality". If all thedata points were situated on the line it would mean that the EC₅₀ valuesdetermined according to the present invention were exactly equal to theLC₅₀ values taken from the literature. Values in FIG. 1 that fall belowthe "line of equality" indicate that the LC₅₀ test was more sensitivethan the EC₅₀ values obtained by the present invention. Values in FIG. 1that fall above the "line of equality" indicate that the presentinvention is a more sensitive test.

FIG. 2 compares Daphnia magna data obtained with the bioassay of thepresent invention with Microtox high and low EC50 pure compound valuestaken from the literature. Microtox is a test available from Microbics,Inc. California, U.S.A. In brief, the Microtox test procedure measuresthe light output of bioluminescent bacteria before and after they arechallenged by a sample of unknown toxicity. The degree of light loss--anindication of metabolic inhibition in the test organism--indicates thedegree of toxicity of the sample. In almost all cases the valuesobtained with the present invention was as sensitive or more sensitivethan the literature results from the Microtox tests. In FIG. 2 each barthat extends above the "0" horizontal line (x axis) indicates that thepresent invention is "x" times more sensitive and for bars extendingbelow the "0" line, the Microtox test is "x" times more sensitive. Forexample, the present invention was between 15 and 200 times moresensitive to CuSO₄ than was Microtox testing.

FIG. 3 compares Daphnia magna sensitivity with 96 hour LC₅₀ purecompound values taken from the literature for Pimephales promelas(fathead minnow). In most cases, the present invention was as sensitiveor more sensitive than the fathead minnow test.

The above methodology has been performed successfully with severalinvertebrate species including Daphnia magna, Daphnia pulex,Ceriodaphnia dubia, Artemia salina and finfish species.

EXAMPLE 3 Determination of Toxicity of Wastestream to Activated Sludge

Treatment plants depend on biological activity in their activated sludgeto degrade pollutants. If the biological life in the activated sludge isadversely affected by toxicants, it can no longer effectively degradepollutants. Whenever a new or altered wastestream is taken into a plantthe biological activity can be adversely affected. The following test isdesigned to address a wastestream's level of toxicity to the plant'sbiological community in the sludge.

A fluorometer (Sequoia-Turner Model 450, available from Sequoia-Turner,Calif., USA) was used to determine the intensity of fluorescence at eachtest concentration. A total of 21 20 ml test tubes with a test volume of15 ml were established with the following proportions of activatedsludge supernatant to test wastestream in triplicate:

    ______________________________________                                        Supernatant        Test Wastestream                                           ______________________________________                                        100% a, b, c        0% a, b, c                                                 50% a, b, c       50% a, b, c                                                 25% a, b, c       75% a, b, c                                                 12% a, b, c       88% a, b, c                                                 6% a, b, c        94% a, b, c                                                 3% a, b, c        97% a, b, c                                                 1.5% a, b, c      98.5% a, b, c                                              ______________________________________                                    

After the test concentrations were mixed, they were allowed to stand for10 minutes before adding 2 mg of 4-Methylumbelliferyl-b-D-galactoside(MUF) to each tube. Each test tube was then rolled between the palms andplaced in a test tube rack. After waiting five minutes (test specific)for the MUF to be altered by the organisms, the intensity of emissionswas determined by exciting the tubes in the fluorometer at 340 and 375nanometers. First, fluorescent determinations were made of all the "a"tubes. Then, "b" tubes and finally the "c" tubes. An EC₅₀ value and itsconfidence limits were determined by conventional methods (See, forexample, EPA/600/4-85/013, supra).

EXAMPLE 4 Determination of the Toxic Effect of Influent on WasteTreatment Sludge

This example was undertaken to determine the toxic effect of an influentto the biologically active sludge at a waste treatment plant as inExample 3.

In this Example, the following wastestream test concentrations were madein triplicate 15 ml test tubes with a test volume of 10 ml: 0, 50, 75and 100% wastestreams. Each of the samples (0, 50 and 75) were dilutedwith autoclaved culture water. To each of the test tubes was pipetted 2ml of a mixed base (MBL). The MBL contains the microbial population. TheMBL was incubated for 1 hour at ambient room temperature. Thereafter,0.4 mg of MUF was added and the test tubes were hand rolled tohomogenize. The microbes were allowed to react with the MUF substratefor 15 minutes. At the end of 15 minutes the tubes were subsampled (3.0ml) and fluorescence was determined by a fluorometer (Sequoia-TurnerModel 450). The fluorescent output from the controls was used as abaseline to judge emissions from test concentrations. EC50 values werecalculated by plotting percent adverse effect against the sampleconcentrations (EPA/600/4-85/013).

EXAMPLE 5 Determination of the LC50 Value and Prediction of the EC50 ofa Plant Discharge

Daphnia magna was exposed to a series of effluent concentrations. Eachtriplicate 10 ml sample was pipetted into a 15 ml glass test tube. Thefollowing effluent test concentrations were assayed: 0% (controls),6.25%, 12.5%, 25%, 50% and 100%. 5 Daphnia magna (±1 day in age) wereadded to each test tube. The test tubes were maintained at ambient roomtemperature for 1 hour. At one hour each tube was injected with 0.4 mgof sonicated MUF in 250 ul of distilled water.

The tubes were then maintained at room temperature for a further periodof 15 minutes. After 15 minutes the series was assessed as in Example 2.From this data, EC₅₀ values were calculated and LC50 values wereaccurately predicted.

Several complex effluents were bioassayed with both conventional 48 hourLC₅₀ methodology and the bioassay of the present invention with Daphniamagna. The results, which are presented in FIG. 4, produced an excellentcorrelation (R² values ranged from 0.90 to 0.96). In FIG. 6, EC₅₀ andLC₅₀ values are expressed in percent effluent.

I claim:
 1. An aquatic bioassay for determining the existence of toxicants in an aquatic source comprising:(a) at least one test sample comprising:i. an aquatic source to be tested; ii. an effective amount of an enzyme substrate selected from the group consisting of 4-Methylumbelliferyl b-D-Galactoside, 4-Methylumbelliferyl a-D-Glucoside, 4--Methylumbelliferyl b-D-Xyloside, 4-Methylumbelliferyl acetate, 4-Methylumbelliferyl N-Acetyl-b-D-Galactosaminide, 4-Methylumbelliferyl N-Acetyl-a-D-Glucosaminide, 4-Methylumbelliferyl-b-D-Glucosaminide, 4-Methylumbelliferyl-a-L-Arabinofuranoside, 4-Methylumbelliferyl-a-L-Arabinoside, 4-Methylumbelliferyl Butyrate, 4-Methylumbelliferyl b-D-Cellobioside, 4-Methylumbelliferyl b-D-N,N'-Diacetyl-Chitobioside, 4-Methylumbelliferyl Elaidate, 4-Methylumbelliferyl b-D-Fucoside, 4-Methylumbelliferyl a-L-Fucoside, 4-Methylumbelliferyl b-L-Fucoside, 4-Methylumbelliferyl a-D-Galactoside, 4-Methylumbelliferyl b-D-Glucoside, 4-Methylumbelliferyl b-D-Glucuronide 4-Methylumbelliferyl Heptanoate, 4-Methylumbelliferyl a-D-Mannopyranoside, 4-Methylumbelliferyl b-D-Mannopyranoside, 4-Methylumbelliferyl Oleate, 4-Methylumbelliferyl Palmitate, 4-Methylumbelliferyl Phosphate, 4-Methylumbelliferyl Propionate, 4-Methylumbelliferyl Stearate, 4-Methylumbelliferyl Sulfate, 4-Methylumbelliferyl b-D-N,N',N"-Triacetyl Chitotriose and 4-Methylumbelliferyl 2,3,5-Tri-O-Benzoyl-a-L-Arabinofuranoside; and iii. a number of multi-cellular organisms having bodies which fluoresce after metabolizing said enzyme substrate; and (b) reference standards, at least one of said reference standards comprising:i. an aquatic source containing a known toxicant concentration; ii. an identical amount and type of said enzyme substrate from (a)ii); and iii. an identical number and type of said living organism from (a)iii); and at least one additional reference standard comprising: i. an aquatic source containing an absence of toxicant; ii. an identical amount and type of said enzyme substrate from (a)ii); and iii. an identical number of organisms from (a)iii).
 2. The bioassay according to claim 1 wherein said enzyme substrate is 4-Methylumbelliferyl-b-D-galactoside in a concentration of about 1 ppm to about 100 ppm.
 3. The bioassay according to claim 1 wherein said bioassay contains at least three reference standards, each reference standard containing different concentrations of toxicant.
 4. The bioassay according to claim 1 wherein said living organisms are selected from the group consisting of, multi-cellular fungi, Cnidaria, members of the phylum Platyhelminthes, molluscs, Polychaetes, members of the phylum Arthropoda, insect larvae, Daphnia spp., juvenile and adult fathead minnows and amphibians.
 5. The bioassay according to claim 1 wherein said living organisms are selected from the group consisting of Daphnia magna, Ceriodaphnia dubia, Daphnia pulex, Daphnia spp., Brachionas spp. (rotifers, Artemia saline (brine shrimp), juvenile and adult fathead minnows, mysid shrimp and fruit fly larvae.
 6. The bioassay according to claim 1 wherein said living organisms are Daphnia magna and said enzyme substrate is 4-Methylumbelliferyl-b-D-galactoside.
 7. A method of determining the toxicity of an aquatic source comprising:(1) preparing a test sample aquatic source; (2) preparing reference samples, each reference sample containing an amount of toxicant ranging form 0 up to at least about the LC₅₀ or EC₅₀ concentration for said toxicant; (3) adding to the samples from steps 1 and 2 a number of multi-cellular organisms having bodies which fluoresce after metabolizing an enzyme substrate selected from the group consisting of 4-Methylumbelliferyl b-D-Galactoside, 4-Methylumbelliferyl a-D-Glucoside, 4-Methylumbelliferyl b-D-Xyloside, 4-Methylumbelliferyl acetate, 4-Methylumbelliferyl N-Acetyl-b-D-Galactosaminide, 4-Methylumbelliferyl N-Acetyl-a-D-Glucosaminide, 4-Methylumbelliferyl-b-D-Glucosaminide, 4-Methylumbelliferyl-a-L-Arabinofuranoside, 4-Methylumbelliferyl-a-L-Arabinoside, 4-Methylumbelliferyl Butyrate, 4-Methylumbelliferyl b-D-Cellobioside, 4-Methylumbelliferyl b-D-N,N'-Diacetyl-Chitobioside, 4-Methylumbelliferyl Elaidate, 4-Methylumbelliferyl b-D-Fucoside, 4-Methylumbelliferyl a-L-Fucoside, 4-Methylumbelliferyl b-L-Fucoside, 4-Methylumbelliferyl a-D-Galactoside, 4-Methylumbelliferyl b-D-Glucoside, 4-Methylumbelliferyl b-D-Glucuronide, 4-Methylumbelliferyl Heptanoate, 4-Methylumbelliferyl a-D-Mannopyranoside, 4-Methylumbelliferyl b-D-Mannopyranoside, 4-Methylumbelliferyl Oleate, 4Methylumbelliferyl Palmitate, 4-Methylumbelliferyl Phosphate, 4-Methylumbelliferyl Propionate, 4-Methylumbelliferyl Stearate, 4-Methylumbelliferyl Sulfate, 4-Methylumbelliferyl b-D-N,N',N"-Triacetyl Chitotriose and 4-Methylumbelliferyl 2,3,5-Tri-O-Benzoyl-a-L-Arabinofuranoside; (4) adding an effective amount of said enzyme substrate to each of the sample from step 3 after incubation and mixing the sample to uniformity; (5) incubating the sample from step 4 at a temperature of about 0° C. to about 30° C. for a period of at least one minute; and (6) exposing the samples from step 5 to an ultraviolet light source and measuring the fluorescent light emitted.
 8. The method according to claim 7 wherein said enzyme substrate is 4-Methylumbelliferyl-b-D-galactoside.
 9. The method according to claim 7 wherein said enzyme substrate is present in a concentration of about 1 ppm to about 100 ppm.
 10. The method according to claim 7 wherein said living organisms are selected from the group consisting of multi-cellular fungi, Cnidaria, members of the phylum Platyhelminthes, molluscs, Polychaetes, members of the phylum Arthropoda, insect larvae, Daphnia spp., fat head minnows and amphibians.
 11. The method according to claim 7 wherein said living organisms are selected from the group consisting of Daphnia magna, Ceriodaphnia dubia, Daphnia pulex, Daphnia spp., Brachionas spp., (rotifers) Artemia saline (brine shrimp), juvenile and adult fat head minnows, mysid shrimp and fruit fly larvae.
 12. The method according to claim 7 wherein said living organism is Daphnia magna and said enzyme substrate is 4-Methylumbelliferyl-b-D-galactoside.
 13. The method according to claim 7 wherein said measuring step is performed by a fluorometer.
 14. The method according to claim 7 wherein said aquatic source is a hatchery and said method is used to determine the health of a fish population in said hatchery.
 15. The method according to claim 7 wherein said aquatic source is a wastestream and said method is used to regulate the rate of introduction of said aquatic source containing said toxicant into a biologically activated waste treatment plant.
 16. The method according to claim 7 wherein said aquatic source is treated water from a waste treatment plant and said method is used to guage the effectiveness of a waste water treatment plant.
 17. A test kit for testing the toxicity of an aquatic source comprising:(a) an enzyme substrate selected from the group consisting of 4-Methylumbelliferyl b-D-Galactoside, 4-Methylumbelliferyl a-D-Glucoside, 4-Methylumbelliferyl b-D-Xyloside, 4-Methylumbelliferyl acetate, 4-Methylumbelliferyl N-Acetyl-b-D-Galactosaminide, 4-Methylumbelliferyl N-Acetyl-a-D-Glucosaminide, 4-Methylumbelliferyl-b-D-Glucosaminide, 4-Methylumbelliferyl-a-L-Arabinofuranoside, 4-Methylumbelliferyl-a-L-Arabinoside, 4-Methylumbelliferyl Butyrate, 4-Methylumbelliferyl b-D-Cellobioside, 4-Methylumbelliferyl b-D-N,N'-Diacetyl-Chitobioside, 4-Methylumbelliferyl Elaidate, 4-Methylumbelliferyl b-D-Fucoside, 4-Methylumbelliferyl a-L-Fucoside, 4-Methylumbelliferyl b-L-Fucoside, 4-Methylumbelliferyl a-D-Galactoside, 4-Methylumbelliferyl b-D-Glucoside, 4-Methylumbelliferyl b-D-Glucuronide, 4-Methylumbelliferyl Heptanoate, 4-Methylumbelliferyl a-D-Mannopyranoside, 4-Methylumbelliferyl b-D-Mannopyranoside, 4-Methylumbelliferyl Oleate, 4-Methylumbelliferyl Palmitate, 4-Methylumbelliferyl Phosphate, 4-Methylumbelliferyl Propionate, 4-Methylumbelliferyl Stearate, 4-Methylumbelliferyl Sulfate, 4-Methylumbelliferyl b-D-N,N',N"-Triacetyl Chitotriose and 4-Methylumbelliferyl 2,3,5-Tri-O-Benzoyl-a-L-Arabinofuranoside in an amount sufficient to produce a final concentration of about 1 ppm to about 100 ppm in each of a test sample and several reference samples; (b) a number of multi-cellular organisms having bodies which fluoresce after metabolizing said enzyme substrate; and (c) directions for using said enzyme substrate and said multi-cellular organism.
 18. The kit according to claim 17 further comprising an exposure chamber.
 19. The kit according to claim 17 further comprising a black (UV) light.
 20. The kit according to claim 17 wherein said living organism is Daphnia magna and said enzyme substrate is 4-Methylumbelliferyl-b-D-galactoside. 